This invention relates to the use of quaternary compounds to stabilize highly-sensitive fluorescent nucleic acid stains in aqueous solvents. This invention also relates to a novel composition for prestained precast agarose electrophoresis gels with increased usable shelf life for high-sensitive nucleic acid detection. This invention further relates to compositions of solvents that are useful as ready-to-use stain solutions.
Fluorescent dyes are widely used for the detection of nucleic acids in biological assays. In particular, a variety of unsymmetrical cyanine dyes have been shown to be highly sensitive and useful in electrophoresis and other solution-based assays for DNA and RNA analysis and detection. Commercial products such as SYBR(copyright) Green I and II stains (Molecular Probes Inc, Eugene OR; U.S. Pat. Nos. 5,436,134 and 5,658,751); GelStar(trademark) stain (BioWhittaker Molecular Applications Inc, Rockland ME, U.S. Pat. No. 5,863,753); and SYBR Gold stain (Molecular Probes, Eugene OR) are some examples of those unsymmetrical cyanine dyes. These dyes provide high sensitivity due to a combination of over 1000-fold fluorescence enhancement upon binding to nucleic acids and very low background in the unbound state. The use of these dyes in ultraviolet trans-illumination enables detection of DNA at picogram levels. The mode of binding of the dyes to nucleic acids is believed to be of a different mechanism than of the more conventional phenanthridinium intercalator dyes such as ethidium bromide and propidium iodide.
Despite the superior sensitivity of unsymmetrical cyanine dyes, these dyes are not stable in aqueous solvents. They are stable in organic solvents such as dimethyl sulfoxide, but must be transferred into aqueous solvents prior to use in biological assays. In aqueous solvents, the sensitivity of these dyes for DNA detection drops to about half within 4 to 14 days of room temperature storage. As a result, precast electrophoresis gels that are prestained with these sensitive stains have a short shelf-life and are not reliably useful, even though precast gels prestained with ethidium bromide are commercially available (e.g. Reliant(copyright) precast gels, sold by BioWhittaker Molecular Applications). Thus, the availability of precast gels with highly sensitive stains is of substantial benefit, especially to high-throughput laboratories, where the large numbers of samples would otherwise necessitate the casting of numerous gels.
Detergents have been used to improve the stability of highly sensitive nucleic acid stains but only marginally prolong the stability of stains, if at all (see Example 1, Table 1), and are not useful for precast gels. Reports from Mathies (Zeng et al 1997, Analytical Biochemistry 252:110-114; Clark et al 1997, Analytical Chemistry 69: 1355-1363) describe the use of tetrapentylammonium ion in TAPS (3-[tris-(hydroxymethyl)methylamino]-1-propanesulfonic acid) buffer to increase the stability of complexes formed between DNA and bisintercalator dyes. However, these reports do not suggest that the stability of the dyes themselves were effected, or mention the effect on unsymmetrical cyanine dyes such as SYBR Green and GelStar stains. Therefore, there is still a need for a method to stabilize highly sensitive nucleic acid stains, and to provide a means of creating prestained, precast gels and solutions with a useful shelf life.
The present invention provides a method for stabilizing highly sensitive fluorescent nucleic acid stains in aqueous solvents by adding one or more quaternary compounds to the solvent. It is also an object of this invention to provide an electrophoresis gel composition for a precast electrophoresis gel system which includes a gel, an electrophoresis buffer, a highly sensitive fluorescent nucleic-acid binding dye, and one or more quaternary compounds, said quaternary compounds causing the dyes incorporated into the gel to have increased stability in aqueous solvents. It is further an object of this invention to provide a stabilized stain solution comprising an aqueous solvent, a fluorescent nucleic acid stain, and a stabilizing amount of one or more quaternary compounds. The quaternary compounds of the invention have the general structural formula R4NX where R4N is a cation and each R is independently a C1-6 alkyl group or a C1-6 alkoxy group, N is nitrogen, X is a halide anion or a hydroxy anion which dissociates from the cation (R4N)+ in an aqueous environment; and wherein the highly sensitive fluorescent nucleic acid stain comprises a cyanine dye. The above objects and advantages of the present invention will be apparent upon consideration of the following detailed description.